The CO dehydrogenase~linked ferredoxin from acetate-grown
Methanosarcina thermophiIa was characterized to determine the structure and
biochemical properties of the iron-sulfur clusters. Chemical and spectroscopic
analyses indicated that the ferredoxin contained two [4Fe-4S] clusters per
monomer of 6,790 Da, although a [3Fe-4S] species was also detected in the
oxidized protein. The midpoint potentials of the [4Fe-4S] and [3Fe~4S] clusters
at pH 7 were -407 m V and + 103 m V, respectively. Evidence from biochemical
and spectroscopic studies indicated that the [3Fe-4S] species may have been
formed from [4Fe-4S] clusters when ferredoxin was oxidized.

The gene encoding the CO dehydrogenase-linked ferredoxin (fdxA) in Ms.
thermophila had the coding capacity for a 6,230-Da protein which contained
eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open
reading frame (ORF1) was also identified which had the potential to encode a
2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from fdxA
and ORF1 were 62% identical. fdxA and ORFI were present as single copies in
the genome and each was transcribed on a monocistronic mRNA. Both fdxA
and ORF1 were transcribed in cells grown on methanol and trimethylamine, but
only the fdxA -specific transcript was detected in acetate-grown cells. The
apparent transcriptional start sites offdxA and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter.

The heterodisulfide of two cofactors unique to the methanogenic
microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell
extracts of Ms. thermophila using electrons from the oxidation of either H2 or
CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced
under the same conditions. The results indicated that methane is formed by
reductive demethylation of CH3-S-CoM using HS-HTP as a reductant in Ms.
thermophila. Coupling of CO oxidation with reduction of the heterodisulfide
suggested that the CO dehydrogenase-linked ferredoxin may be involved,
although the details of electron flow are not known.